Mitochondrial generation of ATP, and the processes which control it, are crucial to nearly every function performed by cells. Uncoupling proteins 2 (UCP2) and 3 (UCP3) are homologues of UCP1 which provide an important level of control over mitochondrial generation of ATP. Accordingly, there has been much interest in discovering the biochemical and physiological function of UCP2 and UCP3. Gain-of-function, overexpression studies have suggested that UCP2 and 3 have uncoupling activity, and when greatly overexpressed in animals, regulate whole body energy metabolism. However, these high level overexpression studies do not provide insight into the function of endogenous levels of UCP2 and 3. Towards these ends, we have created loss- of-function gene knockout mice lacking either UCP2 or UCP3. In the Progress Report section, we provide strong evidence that UCP2 and UCP3 control rates of ATP production. In the case of skeletal muscle, where UCP3 is abundantly expressed, the physiological significance of this is presently being explored. In the case of pancreatic beta-cells, where ATP couples glucose sensing to insulin secretion, this has extremely important implications for regulation of insulin secretion. UCP2 is abundantly expressed in beta-cells. Of note, mice lacking UCP2 have markedly increased glucose-stimulated insulin secretion, demonstrating that UCP2 negatively regulates this process. Of pathophysiologic significance, UCP2 is significantly upregulated in islets of obese mice with inadequate beta-cell function and type 2 diabetes. Importantly, obese mice lacking UCP2 have restored first-phase insulin secretion, increased serum insulin levels and greatly decreased levels of glycemia. These results strongly suggest that UCP2 is a key component of beta-cell glucose sensing, and is a critical link between obesity beta-cell dysfunction and type 2 diabetes. These important issues will be explored in the following Specific Aims: Specific Aim 1: To define the roles of UCP2 and UCP3 in regulating mitochondrial bioenergetics. Specific Aim 2: To determine the mechanism-of-action by which UCP2 negatively regulates insulin secretion. Specific Aim 3: To determine the role of UCP2 in linking obesity to beta-cell dysfunction and type 2 diabetes. Specific Aim 4: To determine the mechanism by which the obese state induces UCP2 expression in islets. These studies should provide major insights into the physiologic and pathophysiologic functions of UCP2 and 3.